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United States Patent |
5,298,262
|
Na
,   et al.
|
March 29, 1994
|
Use of ionic cloud point modifiers to prevent particle aggregation
during sterilization
Abstract
This invention discloses a composition comprised of nanoparticles having a
surface modifier adsorbed on the surface thereof and an anionic or
cationic surfactant as a cloud point modifier associated therewith, which
cloud point modifier is present in an amount sufficient to increase the
cloud point of the surface modifier. A preferred surface modifier is
tyloxapol. Preferred anionic surfactants are dioctylsulfonesuccinate,
sodium dodecyl sulfate and sodium oleate. Preferred cationic surfactants
are dodecyltrimethylammonium bromide and cetrimide tetradecyl ammonium
bromide. This invention further discloses a method of making nanoparticles
having a surface modifier adsorbed on the surface and an anionic or
cationic surfactant as a cloud point modifier associated therewith,
comprised of contacting said nanoparticles with the cloud point modifier
for a time and under conditions sufficient to increase the cloud point of
the surface modifier.
Inventors:
|
Na; George C. (Fort Washington, PA);
Rajagopalan; Natarajan (Phoenixville, PA)
|
Assignee:
|
Sterling Winthrop Inc. (New York, NY)
|
Appl. No.:
|
987904 |
Filed:
|
December 4, 1992 |
Current U.S. Class: |
424/489; 424/487; 424/495; 424/499; 977/DIG.1 |
Intern'l Class: |
A61K 009/14 |
Field of Search: |
424/489,499,495,487
514/78
|
References Cited
U.S. Patent Documents
3272700 | Sep., 1966 | Sluppe | 514/535.
|
4107288 | Aug., 1978 | Oppenheim et al. | 424/499.
|
4540602 | Sep., 1985 | Motoyama et al. | 424/495.
|
4826689 | May., 1989 | Violanto et al. | 424/489.
|
4826821 | May., 1989 | Clements | 514/78.
|
5055288 | Oct., 1991 | Lewis et al. | 424/9.
|
5133908 | Jul., 1992 | Stainmesse et al. | 424/487.
|
5145684 | Sep., 1992 | Liversidge et al. | 424/489.
|
Other References
"Hydrophile-Lipophile Balance and Cloud Points of Nonionic Surfactants"; J.
Pharm Sci., vol. 58, No. 12, Dec. 1969, pp. 1443-1449.
|
Primary Examiner: Page; Thurman K.
Assistant Examiner: Benston, Jr.; William E.
Attorney, Agent or Firm: Davis; William J.
Claims
We claim:
1. A compound comprised of nanoparticles consisting essentially of a
crystalline diagnostic or therapeutic agent and a non-crosslinked nonionic
surfactant adsorbed on the surface thereof and an anionic or cationic
cloud point modifier associated therewith, which cloud point modifier is
present in an amount of 0.005-20% by weight based on the total weight of
the composition and sufficient to increase the cloud point of the surface
modifier, wherein said nanoparticles are resistant to particle size growth
when said composition is heat sterilized at 121.degree. C. for 15 minutes.
2. The composition of claim 1 wherein said nanoparticles contain a
diagnostic agent.
3. The composition of claim 1 wherein said surface modifier is tyloxapol.
4. The composition of claim 2 wherein said diagnostic agent is ethyl
3,5-diacetoamido-2,4,6-triiodobenzoate.
5. The composition of claim 1 wherein said anionic cloud point modifier is
selected from the group cosisting of sodium dodecyl sulfate,
dioctylsulfosuccinate, taurodeoxycholate and sodium oleate.
6. The composition of claim 1 wherein said cationic cloud point modifier is
selected from the group consisting of dodecyltrimethylammonium bromide and
tetradecyl trimethyl ammonium bromide.
7. The composition of claim 1 further comprising an isotonicity maintaining
compound.
8. The composition of claim 7 wherein said maintaining compound is selected
from the group consisting of mannitol or dextrose.
9. The composition of claim 1 further comprising a pH value maintaining
compound.
10. The composition of claim 9 wherein said pH value maintaining compound
is sodium phosphate.
11. The composition of claim 1 in wherein said cloud point modifier
increases the cloud point of said surface modifier above the sterilization
temperature of the nanoparticles.
12. A method for of making nanoparticles having a surface modifier adsorbed
on the surface and an anionic or cationic cloud point modifier associated
therewith, comprised of contacting said nanoparticles with the cloud point
modifier for a time and under conditions sufficient to increase the cloud
point of the surface modifier.
13. The method of claim 12 further comprising the step of sterilizing said
nanoparticle.
14. The method of claim 13 wherein said sterilizing is by steam heat
autoclaving.
15. The composition of claim 1 wherein said nonionic surfactant is selected
from the group consisting of polyoxyethylene alkyl ethers, polyoxyethylene
castor oil derivatives, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene stearates, tyloxapol, poloxamers, and poloxamines.
Description
FIELD OF THE INVENTION
This invention relates to therapeutic and diagnostic compositions with a
modified cloud point, and to a method for the preparation thereof.
BACKGROUND OF THE INVENTION
Nanoparticles, described in U.S. Pat. No. 5,145,684, are particles
consisting of a poorly soluble therapeutic or diagnostic agent onto which
are adsorbed a non-crosslinked surface modifier, and which have an average
particle size of less than about 400 nanometers (nm).
As a result of their small size, sterilization of therapeutic and
diagnostic agents in nanoparticulate form stabilized by a surface modifier
(surfactant) is difficult. Filtration using a filter of 0.22 .mu.m mesh
size is sufficient to remove most bacteria and viruses, but the
nanoparticles, due to their sizes, cannot be sterile filtered.
Conventional autoclaving (steam heat) at 121.degree. C. will result in
substantial aggregation and/or growth of particle size, rendering the
resulting particles unusable.
The aggregation of nanoparticles upon heating is directly related to the
precipitation of the surface modifier (surfactant) at temperatures above
the cloud point of the surfactant where the bound surfactant molecules are
likely to dissociate from the nanoparticles and precipitate, leaving the
nanoparticles unprotected. The unprotected nanoparticles can then
aggregate into clusters of particles. Upon cooling, the surfactant
redissolves into the solution, which then coats the aggregated particles
and prevent them from dissociating into smaller ones.
This invention is directed to novel compositions that allow autoclaving of
nanoparticles with reduced or no particle size growth. These compositions
provide for a modification of the surfactant adsorbed onto nanoparticles
such that the nanoparticles do not agglomerate during autoclaving. This
invention is also directed to a method of making such compositions.
BRIEF SUMMARY OF THE INVENTION
This invention is directed to a composition comprised of nanoparticles
having a surface modifier adsorbed on the surface thereof and an anionic
or cationic surfactant as a cloud point modifier associated therewith,
which cloud point modifier is present in an amount sufficient to increase
the cloud point of the surface modifier.
This invention is further directed to a method of making nanoparticles
having a surface modifier adsorbed on the surface and an anionic or
cationic surfactant as a cloud point modifier associated therewith, said
method comprising contacting said nanoparticles with the cloud point
modifier for a time and under conditions sufficient to increase the cloud
point of the surface modifier.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a composition comprised of nanoparticles
having a surface modifier adsorbed on the surface thereof and an anionic
or cationic surfactant as a cloud point modifier associated therewith,
which cloud point modifier is present in an amount sufficient to increase
the cloud point of the surface modifier. In a preferred embodiment, the
cloud point of the surface modifier is increased above the temperature for
autoclaving of the nanoparticles to prevent agglomeration.
The nanoparticles useful in the practice of this invention include a
surface modifier. Surface modifiers useful herein physically adhere to the
surface of the x-ray contrast agent but do not chemically react with the
agent or itself. Individually adsorbed molecules of the surface modifier
are essentially free of intermolecular crosslinkages. Suitable surface
modifiers can be selected from known organic and inorganic pharmaceutical
excipients such as various polymers, low-molecular weight oligomers,
natural products and surfactants. Preferred surface modifiers include
nonionic and anionic surfactants.
Representative examples of surface modifiers include gelatin, casein,
lecithin (phosphatides), gum acacia, cholesterol, tragacanth, stearic
acid, benzalkonium chloride, calcium stearate, glycerol monostearate,
cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,
polyoxyethylene alkyl ethers, e.g., macrogol ethers such as cetomacrogol
1000, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, e.g., the commercially available Tweens.TM.,
polyethylene glycols, polyoxyethylene stearates, colloidal silicon
dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose
calcium, carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxy propylcellulose,
hydroxypropylmethylcellulose phthlate, noncrystalline cellulose, magnesium
aluminum silicate, triethanolamine, polyvinyl alcohol, and
polyvinylpyrrolidone (PVP). Most of these surface modifiers are known
pharmaceutical excipients and are described in detail in the Handbook of
Pharmaceutical Excipients, published jointly by the American
Pharmaceutical Association and The Pharmaceutical Society of Great
Britain, the Pharmaceutical Press, 1986.
Particularly preferred surface modifiers include polyvinylpyrrolidone,
tyloxapol, poloxamers such as Pluronic.TM. F68 and F108, which are block
copolymers of ethylene oxide and propylene oxide, and poloxamines such as
Tetronic.TM. 908 (also known as Poloxamine 908), which is a
tetrafunctional block copolymer derived from sequential addition of
propylene oxide and ethylene oxide to ethylenediamine, available from
BASF, dextran, lecithin, dialkylesters of sodium sulfosuccinic acid, such
as Aerosol OT.TM., which is a dioctyl ester of sodium sulfosuccinic acid,
available from American Cyanimid, Duponol.TM. P, which is a sodium lauryl
sulfate, available from DuPont, Triton.TM. X-200, which is an alkyl aryl
polyether sulfonate, available from Rohm and Haas, Tween 80, which is a
polyoxyethylene sorbitan fatty acid ester, available from ICI Specialty
Chemicals, and Carbowax.TM. 3350 and 934, which are polyethylene glycols
available from Union Carbide. Surface modifiers which have been found to
be particularly useful include Tetronic 908, the Tweens.TM., Pluronic F-68
and polyvinylpyrrolidone. Other useful surface modifiers include:
decanoyl-N-methylglucamide;
n-decyl .beta.-D-glucopyranoside;
n-decyl .beta.-D-maltopyranoside;
n-dodecyl .beta.-D-glucopyranoside;
n-dodecyl .beta.-D-maltoside;
heptanoyl-N-methylglucamide;
n-heptyl-.beta.-D-glucopyranoside;
n-heptyl .beta.-D-thioglucoside;
n-hexyl .beta.-D-glucopyranoside;
nonanoyl-N-methylglucamide;
n-noyl .beta.-D-glucopyranoside;
octanoyl-N-methylglucamide;
n-octyl-.beta.-D-glucopyranoside;
octyl .beta.-D-thioglucopyranoside; and the like.
A surface modifier useful in the present invention is tyloxapol (a nonionic
liquid polymer of the alkyl aryl polyether alcohol type; also known as
superinone or triton).
This surface modifier is commercially available and/or can be prepared by
techniques known in the art.
The nanoparticles useful in the practice of this invention can be prepared
according to the methods disclosed in U.S. Pat. No. 5,145,684, whose
disclosure is incorporated herein by reference. Briefly, nanoparticles are
prepared by dispersing a poorly soluble therapeutic or diagnostic agent in
a liquid dispersion medium and wet-grinding the agent in the presence of
grinding media to reduce the particle size of the contrast agent to an
effective average particle size of less than about 400 nm. The particles
can be reduced in size in the presence of a surface modifier.
A general procedure for preparing the particles useful in the practice of
this invention follows. The therapeutic or diagnostic agent selected is
obtained commercially and/or prepared by techniques known in the art as
described above, in a conventional coarse form. It is preferred, but not
essential, that the particle size of the coarse therapeutic or diagnostic
substance selected be less than about 100 .mu.m as determined by sieve
analysis. If the coarse particle size of that agent is greater than about
100 .mu.m, then it is preferred that the coarse particles of the
therapeutic or diagnostic agent be reduced in size to less than 100 .mu.m
using a conventional milling method such as airjet or fragmentation
milling.
The coarse therapeutic or diagnostic agent selected can then be added to a
liquid medium in which it is essentially insoluble to form a premix. The
concentration of the therapeutic or diagnostic agent in the liquid medium
can vary from about 0.1-60%, and preferably is from 5-30% (w/w). It is
preferred, but not essential, that the surface modifier be present in the
premix. The concentration of the surface modifier can vary from about 0.1
to 90%, and preferably is 1-75%, more preferably 10-60% and most
preferably 10-30% by weight based on the total combined weight of the drug
substance and surface modifier. The apparent viscosity of the premix
suspension is preferably less than about 1000 centipoise.
The premix can be used directly by wet grinding to reduce the average
particle size in the dispersion to less than 400 nm. It is preferred that
the premix be used directly when a ball mill is used for attrition.
Alternatively, the therapeutic or diagnostic agent and, optionally, the
surface modifier, can be dispersed in the liquid medium using suitable
agitation, e.g., a roller mill or a Cowles type mixer, until a homogeneous
dispersion is observed in which there are no large agglomerates visible to
the naked eye. It is preferred that the premix be subjected to such a
premilling dispersion step when a recirculating media mill is used for
attrition.
Wet grinding can take place in any suitable dispersion mill, including, for
example, a ball mill, an attritor mill, a vibratory mill, and media mills
such as a sand mill and a bead mill. A media mill is preferred due to the
relatively shorter milling time required to provide the intended result,
i.e., the desired reduction in particle size. For media milling, the
apparent viscosity of the pr.mu.mix preferably is from about 100 to about
1000 centipoise. For ball milling, the apparent viscosity of the premix
preferably is from about 1 up to about 100 centipoise. Such ranges tend to
afford an optimal balance between efficient particle fragmentation and
media erosion.
The grinding media for the particle size reduction step can be selected
from rigid media preferably spherical or particulate in form having an
average size less than about 3 mm and, more preferably, less than about 1
mm. Such media desirably can provide the particles of the invention with
shorter processing times and impart less wear to the milling equipment.
The selection of material for the grinding media is not believed to be
critical. However, preferred media have a density greater than about 3
g/cm.sup.3. Zirconium oxide, such as 95% ZrO stabilized with magnesia,
zirconium silicate, and glass grinding media provide particles having
levels of contamination which are believed to be acceptable for the
preparation of therapeutic or diagnostic compositions. However, other
media, such as stainless steel, titania, alumina, and 95% ZrO stabilized
with yttrium, are believed to be useful.
The attrition time can vary widely and depends primarily upon the
particular wet grinding mill selected. For ball mills, processing times of
up to five days or longer may be required. On the other hand, processing
times of less than 1 day (residence times of about one minute up to
several hours) have provided the desired results using a high shear media
mill.
The particles must be reduced in size at a temperature which does not
significantly degrade the therapeutic or diagnostic agent. Processing
temperatures of less than about 30.degree.-40.degree. C. are ordinarily
preferred. If desired, the processing equipment can be cooled with
conventional cooling equipment. The method is conveniently carried out
under conditions of ambient temperature and at processing pressures which
are safe and effective for the milling process. For example, ambient
processing pressures are typical of ball mills, attritor mills and
vibratory mills. Processing pressures up to about 20 psi (1.4 kg/cm.sup.2)
are typical of media milling.
The surface modifier, if not present in the premix, must be added to the
dispersion after attrition in an amount as described for the premix.
Thereafter, the dispersion can be mixed, e.g., by shaking vigorously.
Optionally, the dispersion can be subjected to a sonication step, e.g.,
using an ultrasonic power supply. For example, the dispersion can be
subjected to ultrasonic energy having a frequency of 20-80 kHz for a time
of about 1 to 120 seconds.
The relative amount of therapeutic or diagnostic agent and surface modifier
can vary widely and the optimal amount of the surface modifier can depend,
for example, upon the particular therapeutic or diagnostic agent and
surface modifier selected, the critical micelle concentration of the
surface modifier if it forms micelles, the hydrophilic lipophilic balance
(HLB) of the stabilizer, the melting point of the stabilizer, its water
solubility, the surface tension of water solutions of the stabilizer, etc.
The surface modifier preferably is present in an amount of about 0.1-10 mg
per square meter surface area of the therapeutic or diagnostic agent. The
surface modifier can be present in an amount of 0.1-90%, preferably 1-75%,
more preferably 10-60%, and most preferably 10-30% by weight based on the
total weight of the dry particle.
Therapeutic and diagnostic agents useful in the composition of the present
invention include those disclosed in U.S. Pat. No. 5,145,684 and EP-A
498,482, whose disclosures are incorporated herein by reference. A
preferred diagnostic agent is the x-ray imaging agent WIN-8883 (ethyl
3,5-diacetoamido-2,4,6-triiodobenzoate).
As used herein, particle size refers to a number average particle size as
measured by conventional particle size measuring techniques well known to
those skilled in the art, such as sedimentation field flow fractionation,
photon correlation spectroscopy, or disk centrifugation. By "an effective
average particle size of less than about 400 nm" it is meant that at least
90% of the particles have a weight average particle size of less than
about 400 nm when measured by the above-noted techniques. In preferred
embodiments of the invention, the effective average particle size is less
than about 300 nm, and more preferably less than about 250 nm. In some
embodiments of the invention, an effective average particle size of less
than about 200 nm has been achieved. With reference to the effective
average particle size, it is preferred that at least 95% and, more
preferably, at least 99% of the particles have a particle size less than
the effective average, e.g., 400 nm. In particularly preferred
embodiments, essentially all of the particles have a size less than 400
nm. In some embodiments, essentially all of the particles have a size less
than 250 nm.
A method for the preparation of a nanoparticle composition according to
this invention includes the steps of introducing a therapeutic or
diagnostic agent, a liquid medium, grinding media, and optionally, a
surface modifier into a grinding vessel; wet grinding to reduce the
particle size of the therapeutic or diagnostic agent to less than about
400 nm; and separating the particles and optionally the liquid medium from
the grinding vessel and grinding media, for example, by suction,
filtration or evaporation. If the surface modifier is not present during
wet grinding, it can be admixed with the particles thereafter. The liquid
medium, most often water, can serve as the pharmaceutically acceptable
carrier. The method preferably is carried out under aseptic conditions.
Thereafter, the nanoparticle composition preferably is subjected to a
sterilization process.
As noted elsewhere herein, sterile filtration will not provide adequate
sterilization for nanoparticles. Therefore, other methods of sterilization
are required. For example, steam or moist heat sterilization at
temperatures of about 121.degree. C. for a time period of about 15 minutes
can be used. At altitudes near sea level, such conditions are attained by
using steam at a pressure of 15 pounds per square inch (psi) in excess of
atmospheric pressure.
Dry heat sterilization may also be performed, although the temperatures
used for dry heat sterilization are typically 160.degree. C. for time
periods of 1 to 2 hours.
Sterilization takes place in the presence of ionic cloud point modifiers,
such as an anionic surfactant e.g., sodium dodecyl sulfate (SDS), capronic
acid, caprylic acid, dioctylsulfosuccinate (DOSS), and sodium oleate, or a
cationic surfactant, such as dodecyltrimethylammonium bromide (DTAB) and
tetradecyl trimethyl ammonium bromide, also known as cetrimide (TTAB),
which minimize particle size growth during sterilization.
The cloud point is the temperature at which the surface modifier
(surfactant) precipitates out of solution as described above. By the
phrase "cloud point modifier" is meant a compound which influences the
cloud point of surface modifiers. In particular, the cloud point modifiers
useful in the present invention raise the cloud point of the surface
modifiers found adsorbed onto nanoparticles. In this way, the surface
modifiers do not dissociate from the surface of the nanoparticles at
temperatures used in autoclaving. Therefore, nanoparticles thus modified
do not agglomerate during the sterilization process, and thus retain their
effective average particle sizes of less than about 400 nm after
sterilization.
The ionic cloud point modifier can be present in an amount of 0.005-20%,
preferably 0.01-15%, more preferably 0.05-10%, by weight based on the
total weight of the nanoparticle suspension.
Isotonicity refers to the osmotic pressure of a solution. A solution which
will be administered into the blood stream of an individual is typically
prepared such that the osmotic pressure of that solution is the same as
the osmotic pressure of blood. Such a solution is said to be isotonic.
An isotonicity maintaining compound is a compound which provides for the
maintenance or alteration of a solution so as to make that solution
isotonic. Such an isotonicity maintaining compound will adjust the osmotic
pressure of a solution containing the compositions of the present
invention so as to provide, or maintain, an isotonic solution.
Exemplary isotonicity maintaining compounds include mannitol, dextrose,
sodium chloride, potassium chloride, Ringer's lactate, etc. Preferred
isotonicity maintaining compounds include mannitol and dextrose.
The pH value of a solution is also an important factor. Typically, pH
values should not be either too acidic or too basic. To maintain the pH
value of a solution, it is preferrable to provide pH value maintaining
compounds. These compounds provide a buffering capacity to the solution,
to prevent extremes of pH values of the solution upon storage or upon
subsequent manipulation.
Exemplary pH value maintaining compounds include the well known buffers
such as Tris base, HEPES, carbonate, phosphate, acetate and citrate salts.
A preferred buffer is sodium phosphate (either mono- or di-basic, or
both).
This invention further discloses a method of making nanoparticles having a
surface modifier adsorbed on the surface and an anionic or cationic cloud
point modifier associated therewith, comprised of contacting said
nanoparticles with the cloud point modifier for a time and under
conditions sufficient to increase the cloud point of the surface modifier.
This method involves the preparation of therapeutic or diagnostic
nanoparticles, as discussed elsewhere herein, and contacting those
nanoparticles with an ionic cloud point modifier. Contacting may be by
admixing a suspension of nanoparticles with a solution of cloud point
modifier. In a preferred embodiment, the method is followed by
sterilization at a temperature and for a time sufficient to effect
sterilization of the nanoparticle suspension. A preferred method of
sterilization is steam autoclaving.
The following examples further illustrate the invention and are not to be
construed as limiting of the specification and claims in any way.
EXAMPLE 1: WIN-8883/Tyloxapol formulation
WIN-8883 nanoparticle suspensions are most likely negatively charged.
Therefore, a positively charged surfactant should attach itself very well
to the surface of the particle, as a result of ionic interactions.
WIN-8883 disperses very well in Tyloxapol (3%) solution. However,
Tyloxapol has a very low cloud point (98.degree. C.). To raise the cloud
point, various ionic (both cationic and anionic) cloud point modifiers
were used.
Results of cloud point measurement are shown in Table 1. Neither
polyethylene glycol (PEG-400) nor propylene glycol (PG) is effective in
raising the cloud point of Tyloxapol. Anionic surfactants such as DOSS,
SDS and sodium oleate are very effective in raising the cloud point of
Tyloxapol. The cationic surfactants tested [dodecyl trimethyl ammonium
bromide (DTAB) and tetradecyl trimethyl ammonium bromide (TTAB)] are also
very effective in raising the cloud point of Tyloxapol. Salts such as TRIS
and phosphate lower the cloud point of Tyloxapol, phosphate having a
stronger effect than TRIS.
TABLE 1
______________________________________
Effect of Ionic and Nonionic Additives on the Cloud
Point of Tyloxopol (1%)
Cloud Point
Additive Concentration
(.degree.C.)
______________________________________
Control (none) 94
PEG-400 10% (w/v) 105
5% 100
2% 96
Propylene Glycol
2% 98
SDS 0.5% >131
0.2% >131
0.1% >131
0.05% 127
0.01% 115
DOSS 0.2% >131
0.1% >131
0.05% >131
0.01% 116
Sodium oleate 0.5% >131
0.2% >131
0.05% 123
0.01% 116
DTAB 0.5% >131
0.2% 131
0.1% 122
0.05% 114
TTAB 0.5% >131
0.2% >131
0.1% >131
0.05% >131
0.01% 110
Sodium phosphate, pH 6.5
4 mM 93
10 mM 92
TRIS Buffer, pH 7.5
10 mM 93
Diatrizoic Acid 0.1% 124
0.33% 128
Taurodeoxycholate
0.1% 123
0.2% 129
______________________________________
EXAMPLE 2: Particle size of WIN-8883/Tyloxapol
Results indicate that when formulated with a small amount of ionic
surfactant as a cloud point modifier, either anionic or cationic,
WIN-8883/Tyloxapol nanoparticle suspensions remain unchanged in particle
size after autoclaving at 121.degree. C. for 20 minutes. The results are
shown in Table 2.
The results are consistent with the effect of cloud point modifiers on the
cloud point of Tyloxapol. Those that raised the cloud point (SDS, DOSS,
DTAB, CTAB) showed strong stabilization effect whereas those with little
or no effect on cloud point (PEG) showed no stabilization effect.
Also, it appears that low concentration of buffer, either phosphate or TRIS
can be added without much detrimental effect.
TABLE 2
______________________________________
Stabilizing Effect of Ionic Surfactants on a
Nanoparticle Suspension
(15% WIN 8883/3% Tyloxapol), pH 4.2
Autoclave
Sterilization
Mean Particle
Additive 121.degree. C./20 min.
Size (nm) Polydispersity
______________________________________
none no 158 0.102
none yes 445 0.231
5% PEG-400
yes 453 0.246
10% PEG-400
yes 507 0.197
10% PEG + yes 237 0.134
0.5% DTAB
0.5% DTAB yes 209 0.182
0.3% DTAB yes 245 0.178
0.2% DTAB yes 250 0.179
0.3% TTAB yes 295 0.209
0.5% SDS yes 185 0.115
0.3% SDS yes 188 0.135
0.2% SDS yes 185 0.131
0.1% SDS yes 190 0.134
0.5% DOSS yes 176 0.158
0.3% DOSS yes 190 0.116
0.2% DOSS yes 188 0.136
with 10 mM Sodium Phosphate buffer (pH 6.65)
none yes 406 0.187
0.2% DTAB yes 350 0.117
0.2% DOSS yes 185 0.137
0.1% SDS yes 179 0.155
______________________________________
EXAMPLE 3: Cloud Point Analysis of Tyloxapol
In order to determine the effect of various buffers and various surfactants
on the cloud point of Tyloxapol, the following general methodology was
used. First, using 5 milliliter treated Wheaton vials, the amount of the
additives to be tested were weighed into each vial. Next, 2.0 ml of a 1%
Tyloxapol stock solution was added to each vial. The vials were then
placed in a PEG-400 bath and the temperature was increased slowly to
observe the solution turning cloudy. The results of these experiments are
shown in Table 3.
TABLE 3
______________________________________
Cloud Point Determination of Tyloxapol (1%)
Additive Cloud Point (.degree.C.)
Increase of C.P
______________________________________
None 95 0
10% PEG-400 105 10
5% PEG-400 100 5
0.5% SDS >131 >36
2% Propylene Glycol
98 3
0.2% DTAB 131 36
0.5% DTAB >131 >36
0.5% TTAB >131 >36
0.5% Sodium Oleate
>131 >36
0.5% DOSS >131 >36
______________________________________
EXAMPLE 4: Effect of SDS and DOSS on the particle size of EEDA
nanoparticles
DOSS and SDS samples were prepared by adding specific volumes of DOSS or
SDS stock solution (in 3% Tyloxapol) to nanoparticle solutions as in
Example 3. Samples were autoclaved in the steam autoclave at 121.degree.
C. as indicated in the Table. The results are shown in Table 4.
TABLE 4
______________________________________
Particle Size Analysis of WIN 8883/Tyloxapol
Nanoparticle Suspension
Sample: 15% WIN 8883, 3% Tyloxapol.
Average particle size: 159 nm
Autoclaved at 121.degree. C. for 20 min.
Additive Mean Particle Size (nm)
Poly dispersity
______________________________________
none 445 0.231
0.05% SDS 376 0.1
0.1% SDS 186 0.129
0.04% DOSS 415 0.183
0.1% DOSS 189 0.125
______________________________________
EXAMPLE 5: Effect of Stabilizers and Isotonicity maintaining compound on
the stability of WIN 8883 nanoparticles
The addition of ionic cloud point modifiers and isotonicity maintaining
compounds were tested in nanoparticle suspensions of WIN 8883/Tyloxapol,
as described in Example 3. The results are shown in Table 5.
TABLE 5
______________________________________
Stabilizing Effect of Ionic Surfactants on WIN 8883
nanoparticles upon Autoclave Sterilization (all samples
autoclaved at 121.degree. C. for 20 min.)
Additive Mean Particle Size (nm)
Polydispersity
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Sample: 15% WIN 8883, 3% Tyloxapol, pH 6.0 + 2.5% Glycerol
0.2% DOSS 181 0.22
0.2% DOSS 184 0.17
0.2% SDS 186 0.16
Sample: 15% WIN 8883, 3% Tyloxapol, pH 6.0 + 5% Mannitol
0.2% DOSS 183 0.19
0.2% SDS 186 0.13
Sample: 15% WIN 8883, 3% Tyloxapol, pH 6.0 + 5% Dextrose
0.2% DOSS 182 0.17
0.2% SDS 187 0.18
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Stabilizing Effect of Ionic Surfactants on Particle Size
Distribution
121.degree. C./
Z Ave.
Additive 20 min. (nm) Polydispersity
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Sample: 15% WIN 8883, 0.93% Tyloxapol
control (no additive)
no 1577 0.362
control (no additive)
yes 1275 0.486
0.2% DOSS, 5% mannitol
yes 471 0.31
Sample: 15% WIN 8883, 2% Tyloxapol
control (no additive)
no 158 0.146
control (no additive)
yes 415 0.198
0.2% SDS, 5% mannitol
yes 170 0.142
0.2% DOSS yes 170 0.157
0.2% SDS yes 168 0.083
0.2% SDS, 5% dextrose
yes 170 0.098
0.2% DOSS, 5% mannitol
yes 174 0.085
0.2% DOSS, 5% dextrose
yes 169 0.139
0.1% SDS yes 180 0.139
0.1% DOSS, 5% mannitol
yes 184 0.147
0.1% SDS, 5% mannitol
yes 187 0.135
0.1% DOSS yes 183 0.087
0.1% SDS, 5% dextrose
yes 180 0.159
0.1% DOSS, 5% dextrose
yes 180 0.096
Sample: 15% WIN 8883, 3% Tyloxapol
control (no additive)
no 143 0.06
control (no additive)
yes 452 0.167
0.2% DOSS, 5% dextrose
yes 168 0.138
0.2% SDS, 5% mannitol
yes 169 0.153
0.2% DOSS yes 168 0.108
0.2% SDS, 5% dextrose
yes 169 0.12
0.2% SDS yes 163 0.159
0.2% DOSS, 5% mannitol
yes 169 0.126
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The foregoing specification, including the specific embodiments and
examples is intended to be illustrative of the present invention and is
not to be taken as limiting. Numerous other variations and modifications
can be effected without departing from the true spirit and scope of the
present invention.
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